Effect of Lung Surfactant Protein SP-C and SP-C-Promoted Membrane Fragmentation on Cholesterol Dynamics.

Article Effect of Lung Surfactant Protein SP-C and SP-C-Promoted Membrane Fragmentation on Cholesterol Dynamics Nuria Roldan,1 Thomas K. M. Nyholm,2 J. Peter Slotte,2 Jesús Pérez-Gil,1 and Begoña Garcı́a-Álvarez1,* 1 Department of Biochemistry and Molecular Biology I, Complutense University, Madrid, Spain; and 2Biochemistry, Faculty of Science and Engineering, Åbo Akademi University, Turku, Finland ABSTRACT To allow breathing and prevent alveolar collapse, lung surfactant (LS) develops a complex membranous system at the respiratory surface. LS is defined by a specific protein and lipid composition, including saturated and unsaturated phospholipid species and cholesterol. Surfactant protein C (SP-C) has been suggested to be an essential element for sustaining the presence of cholesterol in surfactant without functional impairment. In this work, we used a fluorescent sterol-partitioning assay to assess the effect of the surfactant proteins SP-B and SP-C on cholesterol distribution in membranes. Our results suggest that in the LS context, the combined action of SP-B and SP-C appears to facilitate cholesterol dynamics, whereas SP-C does not seem to establish a direct interaction with cholesterol that could increase the partition of free cholesterol into membranes. Interestingly, SP-C exhibits a membrane-fragmentation behavior, leading to the conversion of large unilamellar vesicles into highly curved vesicles ~25 nm in diameter. Sterol partition was observed to be sensitive to the bending of bilayers, indicating that the effect of SP-C to mobilize cholesterol could be indirectly associated with SP-C-mediated membrane remodeling. Our results suggest a potential role for SP-C in generating small surfactant structures that may participate in cholesterol mobilization and pulmonary surfactant homeostasis at the alveolar interfaces. INTRODUCTION From the first moment the lungs of a newborn are exposed to air, lung surfactant (LS) complexes secreted by pneumocytes rapidly cover the alveolar air-liquid interface. There, LS layers prevent alveolar collapse by lowering the surface tension to values close to 0 mN/m during expiration, thereby minimizing the work of breathing and allowing the lungs to expand and compress normally (reviewed in (1)). Lipids account for 90% of the LS total mass. The exact proportion of saturated/unsaturated phospholipids and cholesterol is still a matter of discussion, especially with regard to the human lung. However, it is widely accepted that in most mammals, disaturated phospholipids such as dipalmitoylphosphatidylcholine (DPPC) represent ~40% of LS lipids. These saturated phospholipids are responsible for the low surface tension values observed upon expiration. Other PC species are also present in LS, as well as phospholipids such as phosphatidylinositol and phosphatidylglycerol. In LS neutral lipids, cholesterol accounts for ~8–10% by mass of the whole lipid Submitted March 10, 2016, and accepted for publication September 6, 2016. *Correspondence: Editor: Andreas Engel. http://dx.doi.org/10.1016/j.bpj.2016.09.016 fraction (1). Specific surfactant proteins (SP-A, SP-B, SP-C, and SP-D) represent <10% of the surfactant mass but are crucial components for the proper function of LS. Surfactant optimal biophysical function is essentially achieved by the presence of the small hydrophobic polypeptide SP-B, while the transmembrane protein SP-C serves to enhance lipid adsorption and modulate the activity of cholesterol-containing films. SP-B and SP-C each account for <1% of surfactant mass. LS membranes and monolayers have a complex and dynamic lateral structure that is determined by its lipid composition and includes segregation of liquid-ordered (Lo) and -disordered (Ld) fluid phases (2). The proteins SP-B and SP-C do not appear to contribute to lateral phase segregation (2), but they do contribute to the formation of membrane stacks that generate highly cohesive multilayer assemblies (3). However, cholesterol has been shown to be essential for lateral phase segregation, especially at nearphysiological temperatures (2,4). Removal of cholesterol substantially alters phase coexistence in LS membranes (2), but functionally its incorporation above a certain threshold has been reported to be deleterious for the proper surface activity of some clinical surfactant preparations (5). However, it has also been shown that cholesterol improves Ó 2016 Biophysical Society. Biophysical Journal 111, 1703–1713, October 18, 2016 1703 Roldan et al. the spreading capability of model surfactant lipids (6). These contradictory findings suggest that the role of cholesterol is subtly related to the architecture and properties of LS at the air-water interface, and that it could critically depend on the presence of other LS components and/or LS structures assembled in the native complexes. Surfactant protein SP-B is essential for LS function. This protein exists naturally as a covalent dimer supporting the stability of multilayered films at the air-liquid interface. SP-B also improves lipid adsorption and the respreading ability of surfactant material along the breathing cycles (reviewed in (7)). Previous evidence showed that SP-B alone is not enough to sustain the functionality of cholesterol-containing mixtures (8). Remarkably, the presence of the lipopeptide SP-C has been shown to prevent the dysfunction of surfactant preparations incorporating cholesterol (8,9). This small (4.2 kDa) protein increases cholesterol miscibility in surfactant-mimicking membranes (9), and SP-C exposure in bilayers has been reported to be affected by the presence of cholesterol (10). Therefore, SP-C may be an important factor in modulating cholesterol in the LS context, although the molecular mechanisms connecting SP-C and cholesterol remain unclear. Our aim in this work was to obtain further information about the role of the hydrophobic surfactant proteins SP-B and SP-C in cholesterol distribution in LS membranes. Recently, Nyström and co-workers (11) developed a novel fluorescence-based method to determine the effect of transmembrane peptides on the affinity of sterols for membranes. With this technique, it is possible to follow the partitioning of a fluorescent analog of cholesterol (cholesta-5,7,9(11)trien-3-b-ol (CTL)) between membranes and a solubilizing agent such as cyclodextrin by measuring the anisotropy of the fluorophore. These measurements allow the determination of a partition coefficient, Kx, which indicates the affinity of a sterol for a given membrane. Using the CTL partitioning assay, we prepared model and native surfactant membranes in the presence or absence of SP-B and SP-C to assess their effects on the affinity of sterols for such membranes. These surfactant proteins contributed substantially to alter CTL distribution in a specific manner both together and separately. Moreover, our results show that membrane curvature has a significant influence on CTL’s affinit (...truncated)